PIK3CA mutations and their impact on survival outcomes of patients with endometrial cancer: A systematic review and meta-analysis

Several studies have highlighted the frequent alterations of the PI3K pathway in endometrial cancer leading to increased signaling activation with potential for targeted treatment. The objective of this meta-study was to evaluate how PIK3CA exon 9/20 mutations affect survival in endometrial cancer patients, based on available literature. Topic-based search strategies were applied to databases including CENTRAL, MEDLINE, Embase, Web of Science and COSMIC. All studies assessing the impact of mutations in exon 9 and exon 20 of PIK3CA on survival rates of endometrial cancer patients were selected for inclusion. Statistical meta-analysis was performed with the ‘meta’ package in RStudio. Overall, 7 of 612 screened articles were included in the present study, comprising 1098 women with endometrial cancer. Meta-analysis revealed a tendency of impaired survival for patients with PIK3CA exon 9 and/or exon 20 mutations (RR 1.28; 95% CI 0.84, 1.94; p = 0.25). This tendency was consistent in subgroup analyses stratified by histologic type or -grade, with the most prominent effect in low-grade endometrial cancers (RR 2.04; 95% CI 0.90, 4.62; p = 0.09). In summary, these results suggest that PIK3CA mutations negatively influence survival outcomes of patients with endometrial cancer, including those with low-grade tumors.


Introduction
Endometrial cancer (EC) is the most frequent pelvic malignancy among women in countries with a high human developmental index [1]. Obesity and high age are important risk factors for the disease, and with rising obesity rates and an aging world population, the number of patients with EC will increase by over 45% worldwide by 2040, along with the associated mortality [1].
ECs has traditionally been classified into type I or type II based on histological characteristics, grade and hormone receptor expression [2]. As recent molecular studies have further revealed disease heterogeneity, this dualistic classification of EC is now supplemented by four distinct molecular subgroups with disparate prognosis: POLE mutated (ultra-mutated), microsatellite instable (hypermutated), copy-number-low (microsatellite stable) and copy-numberhigh (serous-like) [3]. Accordingly, more accurate diagnosis, prognostic prediction and individualized treatment of EC is further augmented by genomic and molecular studies. Despite this, areas of uncertainty remain, including how mutations in PIK3CA, the second most altered gene in EC, affect patient survival. PIK3CA encodes the p110α protein, the catalytic subunit of the phosphatidylinositol-3-kinase (PI3K) enzyme which is a key component in the PI3K signaling pathway. This pathway is often altered in solid cancers, most commonly in epithelial cancers, with the most frequent aberration rate in EC [4,5]. The pathway is critical in the regulation of cell survival, proliferation and apoptosis, and dysregulation is associated with carcinogenesis [4]. This has made the PI3K pathway an attractive therapeutic target, and evaluation of dual PI3K/mTOR inhibitors, pan-PI3K inhibitors, and isoform-specific inhibitors in combination therapy have shown promising results in clinical trials, also in EC [6][7][8][9][10][11][12].
The reported overall PIK3CA mutation frequency in EC is ranging from 12-39% by Sanger sequencing [13][14][15][16][17][18] and 50% according to next generation sequencing (NGS) [19] with differences likely due to regions covered and sequencing methodologies applied. Mutations of PIK3CA cluster at hotspot sites, and in EC 80% of the mutations are located in the helical (amino acid residues p.E542, p.E545 and p.E546) and kinase domains (amino acid residue p. H1047), corresponding to exon 9 and exon 20, respectively [15]. Most PIK3CA mutations lead to a constitutive active PI3K pathway, but interestingly, the literature is inconsistent with regards to the impact of PIK3CA mutations on patient survival across many cancer types [20][21][22][23][24]. Also in EC, findings are contradictory, and have suggested that PIK3CA mutations are favorable [25], unfavorable [13,18] or have neutral effect on patient survival [13,26,27]. Understanding the role of PIK3CA mutations in relation to clinical survival in endometrial cancer is essential for further development of selective p110a inhibitors, clinical trials and therapeutic approaches. We therefore aimed to investigate the association between PIK3CA exon 9/20 mutations with survival outcomes in patients with EC through a systematic review and meta-analysis.

Materials and methods
The present meta-study was designed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines [28]. The study was based on review of data that have already been published, and therefore patient consent and institutional review board approval were not required. The study is registered with PROSPERO (International Prospective Register of Systematic reviews) ID CRD42021230618.

Types of studies and patients
The eligibility criteria for the inclusion of studies were predetermined. All observational studies (prospective, retrospective and case series) that assessed the impact of mutations in exon 9 and/or exon 20 of the PIK3CA gene on survival rates (disease-specific survival [DSS] and/or overall survival [OS]) of EC patients were selected for inclusion. Studies were selected irrespective of the stage of disease and histological subtype.  [29]. The search employed topic-based strategies designed for each database up to 20 th November 2020, with no rear time limit. Language was restricted to English, with no geographical restrictions. Published conference proceedings that were included in the employed databases were also considered eligible for inclusion but were excluded if final publications were identified. The complete search strategy is presented in Fig 1 and S1 File. Updated literature searches were conducted in all mentioned databases on June 26 th 2022, but none of the newly released papers fulfilled the inclusion criteria.

Data extraction
All records retrieved from the database searches were stored and managed in EndNote reference manager and Rayyan web-tool for systematic reviews [30]. Data were extracted from eligible studies that fitted the inclusion criteria by a stepwise process. First, two reviewers (H.K. B., E.A.H.) independently and blindly reviewed the titles and abstracts of all papers. Secondly, the two authors read the full text of all selected articles and excluded studies that did not meet the inclusion criteria. Thirdly, the two authors independently reviewed and extracted data from the full text articles of included studies. The following data were extracted in a standardized manner: author(s), publication year, country, study design, characteristics of the study population, follow-up time, survival data and PIK3CA mutation data. PIK3CA exon 9/20 mutations were selected for historical reasons with prior available sequencing technologies (mainly Sanger sequencing). In studies using NGS, data were extracted for PIK3CA exon 9/20 mutations only, to avoid bias. In studies where both OS and DSS were reported, data on DSS were extracted rather than OS. At all stages, disagreements were resolved by consensus of the authors or by a third reviewer (C.K.). When data in published papers was missing or not presented clearly, we manually curated data from online databases when possible and/or contacted authors for clarification.

Outcome measure
The outcome measure was predefined during the design of the study as relative risk (RR) of survival rates (overall survival [OS] and/or disease-specific survival [DSS]).

Quality assessment
Risk of bias for each included study was independently assessed by the same initial reviewers (H.K.B. and E.A.H.) using the Newcastle-Ottawa Scale, with the third reviewer (C.K.) mediating in situations of disagreement. The Newcastle-Ottawa Scale examines the risk of bias in observational studies by evaluating the selection of the study groups, the comparability of the groups, and the ascertainment of outcome of interest [31]. The summarized methodological quality of included articles was evaluated as high, medium or low using the scoring algorithm presented in S2 File.

Statistical analysis
Statistical meta-analysis was performed with the R/RStudio version 1.3.959 using the meta package [32]. In a conservative approach, the random-effects model was preferred over the common-effects model since heterogeneity was anticipated, mainly because of study inclusion irrespective of stage of disease and histological subtype. Statistical heterogeneity and assessment of publication bias was assessed using I 2 , Egger's test, Begg's test, Thompson's test and visual interpretation of forest plots [33][34][35]. An I 2 score < 25% was considered as low heterogeneity, an I 2 score between 25-75% as moderate heterogeneity, and an I 2 score > 75% was considered as high heterogeneity [36]. Confidence intervals (CIs) were set at 95%. A two-sided p-value less than 0.05 was considered statistically significant.

Literature search
A flow diagram describing the systematic literature search is given in Fig 1. The detailed search strategy for each database is given in S1 File. Initial searches yielded 1289 entries. After the removal of 680 duplicates, 609 titles and abstracts were screened. Three additional articles were then identified in already known literature, yielding a total of 62 articles that appeared relevant for inclusion. Of these, 55 articles were excluded for the following reasons: no relevant survival data (n = 48), no PIK3CA mutation status (n = 2) and duplicate data (n = 5).

Included articles
This study includes 1098 patients with PIK3CA mutational status and survival data collected from 7 articles (Table 1) [3,13,14,18,27,37,38]. All studies were retrospective in design and investigated mutations of the exon 9 (helical domain) and exon 20 (kinase domain) of PIK3CA. One study reported data on OS while six studies reported DSS [27]. Clinical data including histologic type, grade, tumor size, tumor grade, myometrial infiltration and lymph node positivity and follow-up time are presented in Table 2. According to the criteria of the Newcastle-Ottawa scale (S2 File) [31] three of the studies were of high quality, two of medium quality, and two of low quality (Table 3). Oda et al. (2005) were the first to report survival outcomes of EC patients according to mutation status of the PIK3CA gene [27]. By sequencing exon 9 and -20 they found a mutation frequency of 36% and reported that PIK3CA mutations were more common in tumors with coexisting PTEN mutations. They did not observe significant differences in OS for patients with mutated PIK3CA gene compared to patients with wild type gene (p = 0.44) [27]. Catasus et al. (2008) investigated the clinicopathological impact of PIK3CA mutations and their relationship to other common genetic alterations in a larger series of endometrial adenocarcinomas [14]. The mutation frequency of exon 9 and -20 was 29%. They observed that PIK3CA mutations in exon 20 were associated with adverse prognostic features such as high grade, lymphovascular invasion and deep myometrial infiltration. However, only one of the 32 patients with PIK3CA mutations died from disease during a mean follow-up interval of 3.9 years [14].

Qualitative synthesis of survival outcomes
McIntyre et al. (2013) examined the prognostic significance of PIK3CA mutations in 99 high-grade ECs of both endometrioid and non-endometrioid histology. They observed a statistically shorter DSS for patients with PIK3CA mutations, with the greatest association in grade 3 endometrioid subtypes. They also found that PIK3CA exon 9/20 missense mutations were  [37]. Three of the patients harbored mutations in PIK3CA, corresponding to a mutation frequency of 19%. None of the patients died of the disease during a follow up of 56 months [37]. Bergstrom et al. (2016) analyzed 11 pairs of primary and metastatic endometrial adenocarcinomas to compare mutational concordance between the tumors [38]. They used a semi-conductor based NGS method and found discordant PIK3CA mutation status between primary tumor and metastasis in 4 cases, with loss of mutation in the metastatic lesion in three cases. One of the 11 patients died of the disease during a follow-up of 60 months. This patient had a PIK3CA mutation conserved between the primary tumor and the metastasis. The PIK3CA exon 9/20 mutation frequency of in the primary tumors was 60%, being the highest reported mutation frequency among the included studies [38]. Mjos et al. (2017) sequenced exon 9 and exon 20 of PIK3CA in 280 primary ECs to assess the relationship between clinicopathologic variables and patient survival [13]. The PIK3CA mutation frequency of exon 9 and -20 was 15% in the primary tumors. While PIK3CA mutations generally had no impact on DSS (p = 0.291), exon 9 mutations were associated with poor DSS (p = 0.033 for all PIK3CA exon 9 mutations, p = 0.018 for charge-changing exon 9 mutations) [13]. Analysis of additional 32 metastases paired to the patient series with primary tumors demonstrated concordance between PIK3CA mutations and similar mutational frequencies.
Berger  Their study comprised the highest number of EC patients among the included studies, and extensive clinical annotation and follow-up data were available at www.cBioPortal.org, including molecular subtype [39]. The mutation frequency of exon 9 and -20 was 29%. There was no significant difference in DSS between the PIK3CA mutated and non-mutated groups (p = 0.193) [3].

Quantitative analysis
The effect of PIK3CA mutations on prognosis of patients with EC. A primary metaanalysis of all included studies did not reveal any significant difference in survival outcomes between ECs with mutated and non-mutated PIK3CA (Fig 2; RR 1.28; 95% CI 0.84, 1.94; p = 0.25). The I 2 score of 30% indicated that a moderate proportion of the interstudy variance was due to heterogeneity, as expected from the qualitative synthesis.
Subgroup meta-analyses based on histologic type. Four studies reported sufficient data to perform subgroup analysis on 680 ECs of endometrioid histology (EEC). The pooled RR was 1.45 ( Fig 3A; 95% CI 0.73, 2.88; p = 0.29), with a tendency towards poorer survival for the EEC patient group with mutated PIK3CA, without reaching statistical significance. The I 2 score of 43% suggested moderate heterogeneity in this analysis.
A total of 202 patients with non-endometrioid tumors (NEEC) were available from three studies. The pooled RR was 1.33 ( Fig 3B; 95% CI 0.87, 2.04; p = 0.18) and the null I 2 score indicated no interstudy variance.  Subgroup meta-analyses based on tumor grade. For analysis within low-grade disease, a total of 454 patients from four studies were analyzed. The I 2 score of 0% indicated that no interstudy variance was due to heterogeneity. The pooled RR was 2.04 ( Fig 4A; 95% CI 0.90, 4.62; p = 0.09), suggesting a doubled risk of poor outcome in the PIK3CA mutated group, reaching borderline significance.
Four studies reported data about 532 patients with high grade disease. The pooled RR was 1.23 ( Fig 4B); 95% CI 0.80, 1.89; p = 0.34) and the I 2 score of 45% revealed that a moderate proportion of the interstudy variance was due to heterogeneity.

Estimation of publication bias
Risk of publication bias was evaluated with Egger's test, supplemented by Begg's test and Thompson's test [33][34][35], as multiple tests are recommended when few studies are available in meta-analysis [40]. The result showed no evidence of publication bias in any of the tests (Egger's test; p = 0.70, Begg's test; p = 0.85, Thompson's test; p = 0.73.), supported by visual interpretation of the funnel plot with a symmetrical distribution of studies (S1 Fig) [41]. However, as few studies are available, the power of these methods is low [42].

Discussion
Seven eligible studies with PIK3CA exon 9/20 mutation rates ranging from 19-55% were included in this meta-analysis to assess the impact of PIK3CA mutations on survival outcomes in EC. We specifically focused on mutations of exon 9 and exon 20 in PIK3CA. These regions account for majority of the total PIK3CA mutational burden detected by NGS, including hotspot mutations in the helical-and kinase domains, and have commonly been target regions of PIK3CA in Sanger sequencing approaches.
In our primary analysis including all seven studies, PIK3CA exon 9/20 mutation status had no significant impact on survival outcomes of patients with EC. Hence, the reported PIK3CA mutations in EC, despite a high mutation frequency, do not contribute considerable to the clinicopathologic phenotypes. We note moderate heterogeneity in our qualitative analysis that could be due to differences in the observed PIK3CA mutational status. This was evident in the range of mutation frequencies reported and may be explained by variation in sequencing technology applied, the source of tissue available for DNA isolation, or inter-and intra-cohort differences in FIGO stages and molecular classes. As presented in Table 1, the included studies used DNA extracted from both FFPE and fresh-frozen tissue for DNA sequencing. Fresh frozen tissue remains the preferred source for DNA, as artifacts may arise from the FFPE-fixation procedure. Concerning FIGO staging, all studies included ECs of all stages, but data were insufficient to stratify PIK3CA mutation status according to FIGO stage. Altogether, the variations in the eligible studies appear to explain our observed moderate heterogeneity in the primary analysis, motivating additional subgroup analyses.
Subgroup analyses were performed for histologic type and grade with no significant association of PIK3CA exon 9/20 mutations with survival. The null I 2 scores in low-grade and NEEC subgroup-analysis show low interstudy heterogeneity, strengthening the confidence of these results.
All our analysis, both primary and subgroup analysis, indicated a tendency (non-significant) of reduced survival for patients with mutated PIK3CA. The strongest effect was observed in the subgroup analysis of low-grade ECs with border line significance (p = 0.09). This could suggest that the effect of exon9/20 PIK3CA mutations on survival depends on the mutational context, possibly influenced by factors such as co-mutations in other genes. The effect of PIK3CA mutations may be more potent in low-grade tumors because these tumors usually have a higher mutation burden with increased potential for synergistic effects, e.g. multimechanistic PI3K activation [43], whereas PIK3CA mutations may be less potent in highgrade ECs where survival already is poorer and the effect may be overpowered by features such as copy-number alterations [3].
The location of the PIK3CA mutation may have clinical relevance. In the study by Catasus et al., PIK3CA exon 9 mutations were more common in low-grade tumors while exon 20 mutations more common in high-grade tumors [14]. Other studies have suggested that there are differences in patient survival depending on the exon-specific location of the PIK3CA mutations, as both individual helical (exon 9) or kinase domain (exon 20) mutations have been associated with adverse prognostic effects in EC [13,14]. In vitro experiments have demonstrated that mutations in exons 9 or exon 20 can transform cancer cells through different and independent signaling pathways [44]. Hotspot mutations in the helical domain of exon 9 affects a surface patch of the p110α protein and changes its ability to interact with other regulatory proteins, whereas hotspot mutations in exon 20 affect the kinase domain in proximity to the hinge region of the catalytic loop leading to constitutive activation [14,45]. Among the studies included in this report, PIK3CA exon 20 mutations were more frequent than exon 9 mutations in four of the studies [13,18,27,38], evenly distributed in two of the studies [3,14] and with lower occurrence in one study [37]. Interestingly, the two studies reporting evenly distributed mutation frequencies between exon 9 and exon 20 were the two studies suggesting favorable outcome for the mutated genotype in the primary analysis [3,14]. Hence, stratifying patients according to the location of PIK3CA mutations in larger cohorts is suggested to potentially reveal underlying effects on survival. Unfortunately, individual patient data regarding location of mutations, FIGO stages and the recent new molecular classification (as suggested by TCGA) was insufficient to perform such subgroup analyses in this study.
Forthcoming studies should include full length mutation data and complete clinical annotations to facilitate proper patient stratification and to elucidate the role of PIK3CA in survival. Such data should also include race/ethnicity for the evaluation of differences in PIK3CA mutation rates that has been observed in EC and that might be relevant for optimal patient treatment in different countries [46].
There are some limitations that dampens the conclusions from our meta-analysis. First, the number of studies that assessed the relationship between PIK3CA mutations and survival of patients with EC was limited, with only seven studies available. Second, the size of the study populations varied considerably among the different studies. Small studies with few patients are often a source of publication bias and are more likely to introduce confounding results than larger studies as this reduces the statistical power of the pooled results [47]. However, the present study also has several important strengths. Risk of publication bias was evaluated using three independent tests where none showed evidence of publication bias. In addition, none of our meta-analyses was interpreted as high level of heterogeneity; the primary analysis was evaluated to intermediate heterogeneity, while the subgroup analysis was evaluated as low-intermediate (NEEC and Grade Low ; low heterogeneity, EEC and Grade High ; intermediate heterogeneity). In addition, assessment of the qualities of studies included in the meta-analysis using the Newcastle-Ottawa scale revealed that our analyses in large are based on papers of sufficient quality. To our knowledge, this study is the most powerful and comprehensive synthesis of evidence on this issue to date with a total sample size of 1098 patients.

Conclusions
In summary, this meta-analysis based on current literature indicates a tendency that PIK3CA mutations in exon 9 and/or exon 20 have a negative effect on survival of patients with EC. This tendency was most prominent in patients with low-grade tumors, suggesting that these patients may favor PIK3CA testing and potential targeted therapy the most.

Statement of ethics
This systematic review leverages on literature already published as individual studies. Survival data were extracted from publications as anonymous data, and hence no institutional review board approval was required.